Combating Drug Resistance in HIV protease: A Structural Approach HIV-1 protease is the target of the most potent anti-viral drugs for the treatment of HIV-1 infection. All of these drugs are the consequence of structure-based drug design. Unfortunately, many viable drug resistant variants of HIV-1 protease have evolved under the selective pressure of drug therapy. To develop an effective therapy, this ensemble of HIV-1 protease variants becomes the therapeutic target rather than one wild-type protease clone. Drug resistance at the molecular level is a subtle change in the balance of recognition events between the relative affinity of the enzyme to bind inhibitors and its ability to bind and cleave substrates. Mutations accumulate and confer drug resistance to HIV-1 protease in a complex interdependent manner that maintains or accentuates viral fitness. At a molecular level these mutations impact inhibitor binding by changing the equilibrium between the unliganded and inhibited forms of protease. These changes can directly alter the active site cavity, such as those primary sites of resistance that occur where the inhibitors protrude from the substrate envelope. Alternatively, the changes can be indirect that occur outside the active site and may affect the flexibility or stability of the unliganded protease. In either case, the effects of mutations are often not additive;the impact of a mutation at one site influences the effect of a mutation at another site, in an interdependent fashion. In this proposal we will collaboratively pursue two overall goals: (1) test the "substrate envelope" hypothesis by designing, synthesizing and assaying novel HIV-1 protease inhibitors that fit within the substrate envelope and ascertain if they are more robust to drug resistant variants of HIV-1 protease (Tidor, Rana and Schiffer labs). (2) elucidate the role of known active and non-active site mutations in conferring resistance to current and new protease inhibitors (Shafer, Swanstrom and Schiffer labs). Specifically our group collects and analyzes structural, thermodynamic and dynamic data to elucidate changes in variant proteases and in inhibitor recognition.